This invention relates to biological assays. In particular, the invention is directed to materials and methods of hybridizing microarrays of nucleic acid molecules for analytical, therapeutic and diagnostic purposes.
Microarrays of DNA or RNA polynucleotides or oligonucleotides are state-of-the-art biological tools used in the investigation and evaluation of genes for analytical, diagnostic, and therapeutic purposes. Microarrays typically comprise a plurality of oligomers, synthesized or deposited on a glass support or substrate in an array pattern. The support-bound oligomers are called xe2x80x9cprobesxe2x80x9d and function to bind or hybridize with a sample of DNA or RNA material under test, called a xe2x80x9ctargetxe2x80x9d in hybridization experiments. However, some investigators bind the target sample under test to the microarray substrate and put the oligomer probes in solution for hybridization. Moreover, some investigators use the reverse definition, referring to the surface-bound oligonucleotides as targets and the solution sample of nucleic acids as probes. Either of the xe2x80x9ctargetsxe2x80x9d or xe2x80x9cprobesxe2x80x9d may be the one that is to be evaluated by the other (thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other). All of these iterations are within the scope of this discussion herein. In use, the microarray surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. The targets are typically labeled with an optically detectable label, such as a fluorescent tag, so that the hybridized targets and probes are detectable with scanning equipment. DNA array technology offers the potential of using a multitude (hundreds of thousands) of different oligonucleotides to analyze changing mRNA populations.
There are numerous types of substrates used in hybridization assays. Common substrates or supports used for microarray assays are siliceous substrates, such as glass. The surface of the substrates are typically treated or derivatized to facilitate binding of the probes to the substrate. For in situ synthesis of probes, the first monomers of the oligomer probe sequences are attached to the substrate surface that is derivatized with a silane or other compounds known in the art to facilitate the bonding of the first monomers. Subsequent monomers are added directly to the monomers of the growing oligomer chain. For deposition of presynthesized or whole probes, such as cDNA probes, the probe is attached to a polymer adsorbed or coated on the surface of the substrate to facilitate bonding. The adsorbed polymer is coated and dried on the substrate surface. The substrate surface derivatizations enable and facilitate the attachment of nucleic acids to the surface of microarray substrates for the manufacture of the microarrays. Surface treatments or derivatization techniques, including those mentioned above, are well known in the art.
Microarrays of oligomer probes, such as oligonucleotides or polynucleotides, are hybridized using conventional methods and hybridization solutions. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ed. 2nd, 1989, vol. 1-3, incorporated herein by reference, describe the considerations and conditions for hybridization of oligonucleotide probes. Probe length, hybridization temperature, as well as other factors that are well known in the art affect hybridization conditions. Typically, hybridizations using synthetic oligomers are usually carried out under conditions that are 5-10xc2x0 C. below the calculated melting temperature Tm of a perfect hybrid to minimize mismatched or non-Watson/Crick base pairing between the probe and target, and maximize the rate at which Watson/Crick base pairs form. Other factors influencing the rate of hybrid formation include the salt concentration, the presence of surfactants, solvents or co-solvents, the concentration of nucleic acid in solution, the length of hybridization, and the degree and method of agitation.
The hybridization solution typically comprises a salt (monovalent cation), either SSPE or SSC buffer that provides buffering capacity between pH 6.8-8.5 (more typically between pH 7.0-7.5), a divalent cation chelating agent (e.g. ethylenediaminetetraacetic acid, EDTA), and agents for blocking non-specific binding of targets to the array surface (surfactants, proteins and/or carrier DNA from an organism unrelated to the experiment at hand). More specifically, a typical hybridization solution contains 6xc3x97SSPE (0.9 M NaCl, 60 mM sodium phosphate (pH 7.4); 6 mM EDTA); or 6xc3x97SSC (0.9 M NaCl, 90 mM sodium citrate (pH 7.0)), 0.5% w/v sodium dodecyl sulfate (SDS); 100 xcexcg/ml denatured, fragmented salmon sperm DNA; and 0.1% nonfat dried milk.
The microarray is hybridized for a period of time ranging from about 2 hours to about 2 days, depending on the make-up of the probes (i.e., probe length and diversity of probe composition) and the complexity of the target, for example, at a controlled temperature, which typically ranges from 20xc2x0 C. to 70xc2x0 C., depending on the melting temperature Tm, as discussed above. Low temperature hybridizations are performed at about 20xc2x0 C. to about 50xc2x0 C. (typically about 37-45xc2x0 C.). High temperature hybridizations are performed at or around 55xc2x0 C. to about 70xc2x0 C. (typically 60xc2x0 C. to 65xc2x0 C.). However, for most nucleic acid microarrays, high temperature hybridizations are preferred in the art since the higher temperature maximizes the rate of Watson/Crick base pairing of nucleotides, while low temperature hybridizations typically maximize Watson/Crick base pairings by use of a co-solvent to lower the Tm. The typical time period for hybridization of a microarray is overnight or longer (i.e., anywhere from 8 hours to 24 hours) so as to hybridize the target. The array is then washed and dried and optically scanned to measure the degree of hybridization using conventional methods and equipment that are well known in the art.
A problem in the DNA microarray hybridization art is sporadic poor hybridization assay performance characterized by low-intensity or missing features on the microarray substrate, high backgrounds, and visually xe2x80x9cblotchyxe2x80x9d substrates. For microarrays containing DNA on adsorbed polymer substrate surfaces, this problem has been observed using conventional hybridization conditions, such as using a solution comprising 20xc3x97SSC (3.0 M NaCl, 300 mM Sodium Citrate (pH 7.0), 10% SDS) at high hybridization temperature of about 65xc2x0 C. and within conventional hybridization times of about 6 hrs. to about 24 hours.
Thus, it would be advantageous to have materials, conditions and methods of hybridizing arrays of oligomers on siliceous substrates that have been treated or coated with a surface adsorbed polymer in biological assays at the preferred higher hybridization temperature range and longer hybridization times without affecting the hybridization assay performance.
The present invention provides a buffer composition and method for hybridizing nucleic acid microarrays with other nucleic acid materials used in high throughput analytical, therapeutic, and diagnostic applications. The method uses an envelope of hybridization conditions for performing assays at high hybridization temperatures for long periods of time. The hybridization conditions of the method advantageously are compatible with siliceous substrates having adsorbed polymer surfaces. The buffer composition and method of the present invention work particularly well on adsorbed polycationic polymer coated siliceous surfaces. The envelope of conditions addresses solution pH and buffer type, salt composition, surfactant composition, temperature and time. The present invention allows sensitive, selective detection of nucleic acid targets, while preserving the intactness of the adsorbed polymer siliceous surface. The buffer composition and method of the invention overcome the problems found in the art by reducing delamination of the surface polymer and reducing residues when performing assays on adsorbed polymer siliceous substrate surfaces at high hybridization temperatures between about 55xc2x0 C. and 70xc2x0 C. for time periods used to hybridize a target material. The buffer composition and method of the present invention provide optimized hybridization assay performance by maintaining the integrity of the adsorbed polymer surface of the siliceous substrates.
In one aspect of the invention, a buffer composition for hybridization assays of arrays of oligonucleotides bound to an adsorbed polymer substrate surface with another nucleic acid material is provided. The buffer composition comprises a pH in a range of pH 6.4 to 7.5, a monovalent cation having a monovalent cation concentration ranging from about 0.01 M to 2.0 M, and a non-chelating buffering agent that maintains the pH within the pH range. The buffer composition may optionally further comprise relatively lower concentrations of an ionic surfactant and/or a chelating agent. The buffer composition has a total cation concentration ranging from about 0.02 M to about 2.0 M, accounting for all components in the buffer solution. In one embodiment, the buffer composition further comprises nucleic acids in solution and is used in a hybridization assay of a nucleic acid material under test that is attached to an adsorbed polymer surface of a siliceous substrate.
In another aspect of the invention, a method of hybridizing a microarray of oligonucleotides on an adsorbed polymer surface of a siliceous substrate with another nucleic acid material is provided. The method of hybridizing comprises the step of incubating the nucleic acid material with the microarray of oligonucleotides on the adsorbed polymer surface in a hybridization solution at a hybridization temperature ranging from about 55xc2x0 C. to about 70xc2x0 C. so as to hybridize the nucleic acid material, wherein the hybridization solution comprises a buffer composition that comprises a pH within a range of pH 6.4 and 7.5, a non-chelating buffering agent, and a monovalent cation in a monovalent cation concentration ranging from about 0.01 M to about 2.0 M. The buffer composition may optionally further comprise relatively lower concentrations of a chelating agent and/or an ionic surfactant.
In still another aspect of the invention, a buffer kit for performing hybridization assays on siliceous substrates having an adsorbed polymer on the substrate surface is provided. In one embodiment, the kit comprises the buffer composition of the present invention that comprises a non-chelating buffering agent, a pH within a range of pH 6.4 and 7.5 and a total cation concentration ranging from about 0.02 M to about 2.0 M. In another embodiment, the kit comprises a components list for the buffer composition of the present invention. The components list comprises the components of the buffer composition of the present invention described above. Optional instructions may be included in the kits. The optional instructions comprise the method of the present invention described above, or instructions for using the buffer composition in a hybridization assay of a nucleic acid material with a siliceous substrate that has an adsorbed polymer surface for attaching to nucleic acids. In still another embodiment, the kit may further a nucleic acid sample for use as a hybridization assay control. Still further, the buffer composition in the kit may further comprise nucleic acids in solution, or the kit may further comprise a microarray of nucleic acids attached to an adsorbed polymer surface of a siliceous array substrate for use with a nucleic acid material in a hybridization assay.
The buffer composition, method and kit of the present invention overcome the problems in the art by reducing degradation of the adsorbed polymer surface of the microarray substrate and any further etching of the substrate, both of which impact the hybridization results between nucleic acid probes and target materials at high temperatures. Further, the present invention does not affect the signal intensities of the fluorescent labeling system used in conventional hybridization assays. The hybridization conditions and methods of the present invention are particularly useful in DNA or RNA microarray assays performed at high temperatures, i.e., above about 55xc2x0 C., and for longer hybridization times, i.e., equal to or greater than about 6 hours, where conventional buffer compositions, including but not limited to SSC and SSPE react with the conventional adsorbed polymer surface of siliceous substrates, and affect hybridization results.